The events involved in rho-dependent transcription termination will be probed in vitro using components (DNA, RNA polymerase, RNA and rho) purified from Escherichia coli K-12 and appropriate mutants. Kinetics, and influence of each component on various aspects of the reaction will be examined, taking advantage of mutant proteins known to be affected in a specific step, e.g., the rho-115 rho that is inactivated by exposure to RNA will be compared with wild-type in ability to retain RNA on a nitrocellulose filter. The ability of the rho-RNA complex will be examined. The process of RNA release and the influence of RNA polymerase on this process will be investigated. Nucleic acids associated with RNA polymerase purified from rho-115 cells will be purified to determine the nature of this material (RNA and DNA content, extent of secondary structure, base composition, sequence from the 3'-OH terminus). The requirements for release of this material from polymerase will be examined. Possible factors involved in release include wild-type rho protein, salt and temperature etc. Experiments are described that may support the notion of a rho-induced change in conformation of RNA polymerase. A genetic complementation system using the tetracycline-resistance transposon Tn10 inserted in the ilv operon to transfer rho alleles will be used to determine dominance and the number of complementation groups formed by rho mutations. This study is particularly pertinent because of the pleiotropic phenotypes associated with rho mutations. These data may suggest a relationship between functional barriers imposed by particular rho mutations and complementation mapping within the rho region. Studies will be initiated to determine the number of proteins that bind rho protein, using anti-rho antibody, and examining extracts of cells grown in a variety of conditions. These experiments may yield a class of regulatory proteins that exert their control by anti-termination. Another set of experiments will be performed to determine if rho mutations effect the level of enzymes involved in nitrogen metabolism, particularly glutamine synthetase.